The present invention relates to an absicisic acid (ABA) signalling component, nucleic acid encoding the signalling component, and processes employing the same.
The plant phytohormone ABA plays an important role in growth and development of plants. In seeds it helps in embryogenesis and formation of seed-storage proteins. It prevents premature germination and growth of many seeds and buds. In vegetative tissue ABA protects the plant against adverse environmental conditions such as drought, high salinity, cold temperature or frost. Many of the actions of ABA result in rather long-term physiological changes and appear mainly to involve modification of gene expression at transcriptional level. Over 150 ABA responsive genes have been isolated from various plant species.
The importance of ABA as a stress hormone in connection with a lack of water was first suggested in 1969 by STC Wright and RWP Hiron at Wye College, London University. They found that ABA content of wheat leaves rose by a factor of 40 during the first half hour of wilting. Similar rise in ABA is now detected in leaves of monocot as well as dicot plants. Application of ABA to leaves causes stomatal closing or inhibits stomatal opening as observed in numerous species. Therefore, during water stress, increased ABA-levels reduce water loss.
We have found a novel protein that mediates in signalling evoked by ABA, and which responds to ABA.
According to the present invention there is now provided a protein capable of affecting an ABA response and comprising one or more of the following:
(i) a hydrophobic C-terminus;
(ii) at least one coiled coil region;
(iii) an EF-hand consensus sequence;
(iv) a nucleotide binding site; and
(v) a hydrophilic N-terminus;
or a variant thereof.
In a preferred embodiment, the protein has (i), (ii), (iii) and (iv) as defined above, and optionally (v).
Further, the protein may also be capable of being cleaved by botulinum C. Thus preferably the protein includes two recognition domains for botulinum C and/or a cleavage site.
Preferably at least one of the coiled coil regions corresponds to an epimorphin pattern.
Preferably there are three coiled coil regions.
Preferably there are three coiled coil regions, at least one of which corresponds to an epimorphin pattern.
Preferably the protein further comprises phosphorylation sites.
The protein may be described as a novel syntaxin (t-SNARE) homolog. At the cellular level, ABA is best characterised by its action in regulating K+ ands Clxe2x88x92 channels at the plasma membrane of stomatal guard cells, leading to stomatal closure that reduces transpirational water loss from leaf. The protein of the present invention may be a membrane-anchored protein that is associated with the plasma membrane. In vivo, both cleavage of the protein by Botulinum C toxin and competition by a soluble C-truncated fragment of the protein have been found to prevent ABA action in controlling K+ and Clxe2x88x92 channels in the guard cells. These, and additional results, show that the protein of the present invention is involved in an ABA signalling complex, and responds to ABA.
The features of the protein can be defined as follows with reference to SEQ ID NO:24
In an especially preferred embodiment, the protein comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4.
The present invention includes variants of the protein defined above. Such variants include proteins having 50% or more overall homology with the sequence of SEQ ID NO:2. Typically the homology is 60% or more, more typically 65%, preferably 70%, more preferably 75%, even more preferably 80% or 85%, especially preferred are 90%, 95%, 98% or 99% homology.
Percentage homology preferably is calculated on the basis of amino acids that are identical in corresponding positions in the two sequences under consideration. Conservative substitutions are not taken into account. In calculation of percentage homology of a putative protein under investigation with the SEQ ID NO:2 or SEQ ID NO:4, if the protein under investigation has a different length, then the calculation is based on the amino acids in the portion of the molecule under investigation that overlaps with the sequence shown in SEQ ID NO:2 or SEQ ID NO: 4.
In particular, the term xe2x80x9chomologyxe2x80x9d as used herein may be equated with the term xe2x80x9cidentityxe2x80x9d.
Here, sequence homology can be determined by a simple xe2x80x9ceyeballxe2x80x9d comparison of any one or more of the sequences with another sequence to see if that other sequence has at least 75% identity to the sequence(s).
Relative sequence homology (i.e. sequence identity) can also be determined by commercially available computer programs that can calculate % homology between two or more sequences. A typical example of such a computer program is CLUSTAL.
Sequence homology (or identity) may moreover be determined using any suitable homology algorithm, using for example default parameters. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail in, for example: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990) xe2x80x9cBasic local alignment search tool.xe2x80x9d J. Mol. Biol. 215:403-410; Madden, T. L., Tatusov, R. L. and Zhang, J. (1996) xe2x80x9cApplications of network BLAST serverxe2x80x9d Meth. Enzymol. 266:131-141.; Gish, W. and States, D. J. (1993) xe2x80x9cIdentification of protein coding regions by database similarity search.xe2x80x9d Nature Genet. 3:266-272; Altschul, S. F., Madden, T. L., Schxc3xa4xc3xa4ffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. (1997) xe2x80x9cGapped BLAST and PSI-BLAST: a new generation of protein database search programs.xe2x80x9d Nucleic Acids Res. 25:3389-3402; Karlin, S. and Altschul, S. F. (1990) xe2x80x9cMethods for assessing the statistical significance of molecular sequence features by using general scoring schemes.xe2x80x9d Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S. and Altschul, S. F. (1993) xe2x80x9cApplications and statistics for multiple high-scoring segments in molecular sequences.xe2x80x9d Proc. Natl. Acad. Sci. USA 90:5873-5877, which are incorporated herein by reference. The search parameters are defined as follows, and are advantageously set to the defined default parameters.
Advantageously, xe2x80x9csubstantial homologyxe2x80x9d when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul with a few enhancements.
Analysis may also be carried out using Lasergene DNA, Madison, U.S.A.
It will be appreciated that proteins capable of affecting an ABA response and/or responding to ABA from other species will exhibit inter-species differences, for example, differences in protein length, amino acid sequence and carbohydrate modifications. There may, for example, be variations in the C- and/or N-terminal residues, and in molecular weight.
In a general sense the term xe2x80x9cvariantxe2x80x9d includes a protein which retains the essential properties, in the present case the ability to affect an ABA response and/or respond to ABA. Variants include allelic variants, and proteins, which differ by conservative amino acid changes. The variants may be natural or non-naturally occurring variants made, for example, by mutagenesis.
By conservative amino acid changes we mean replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methionine and vice versa.
Confirmation that a protein is one which is capable of affecting an ABA response may be made by consideration of sequence homology and/or consideration of its structural relationship.
In addition to full-length proteins, the present invention also encompasses molecules that comprise less than a full length sequence. Such molecules or fragments may be polypeptides or peptides. For use as a substitute, a fragment should retain one or more of the biological activities of the parent molecule.
One method that may be used to test a putative molecule for the ability to affect an ABA response is the use of Xenopus laevis oocycles as a heterologous expression as described below.
The present invention does not cover the native protein according to the present invention when it is in its native environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment. The protein of the present invention may also be isolated in the sense that it is substantially free from other proteins with which it is ordinarily associated. In addition the present invention does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment.
Although in the specific non-limiting example described below the ABA signalling component was obtained from Nicotiana tabacum (tobacco) the present invention relates in general to an ABA signalling component. For example, ABA signalling components from dicotyledonous and monocotyledonous plants, including cereals such as wheat, barley, rice, maize and sorghum; field crops other than tobacco such as canola, sunflower, sugarbeet and cotton; fruit and vegetables. As an example, the corresponding ABA signalling component in maize may be found using reverse transcription followed by polymerase chain reaction (RT-PCR) using known techniques and primers devised from the sequences of SEQ ID NO: 1. Confirmation that an ABA signalling component has been arrived at can be achieved using the assays of the present invention and described herein.
Using this approach we have also determined the corresponding ABA signalling component from Arabidopsis thaliana. The nucleic acid sequence is shown as SEQ ID NO:3 and corresponding amino acid sequence as SEQ ID NO:4. The present invention also includes variants of these sequences defined herein. For the avoidance of doubt the terms xe2x80x9ca protein capable of affecting an ABA responsexe2x80x9d and xe2x80x9cABA signalling componentxe2x80x9d are used interchangeably. The terms refer to a molecule involved in the signal transduction pathway of the hormone abscisic acid. Similarly in relation to the assay method of the invention xe2x80x9csignalling componentxe2x80x9d refers to a molecule involved in the signal transduction pathway of a hormone.
The protein of the present invention may be used in screens to detect protein-protein interactions. In particular, the protein may be used to screen for other members of a signal transduction pathway. One suitable method is the so-called two-hybrid system, in which the DNA-binding domain of the GAL4 protein is fused to the protein of the present invention. A second plasmid is constructed comprising the activation domain of the GAL4 protein fused to a protein (or peptide or polypeptide) under investigation. Interaction between the protein of the present invention and the protein under investigation leads to transcriptional activation of a reporter gene, such as detection by expression of a GAL1-lacZ gene fusion.
Thus the present invention also provides fusion proteins comprising the protein of the present invention, and similarly protein/nucleic acid complexes comprising the protein of the present invention. The protein and/or nucleic acid of the present invention may be associated with, for example, a targeting sequence, such a sequence which targets the nucleic acid encoding for the protein to a cell membrane.
Thus, the present invention also provides a method of detecting proteins which interact with the present protein. Interactive proteins found using such a screen are also the subject of the present invention. For example, we have found that there is interaction between the SYR protein of the present invention (see below) and clone 4 (see table 1) which is identified as having a phosphatase inhibitor domain.
Also included within the present invention are truncated proteins derivable from the proteins defined above. Typically such truncated proteins will be able to compete with the non-truncated protein in an ABA signalling pathway, and/or be capable of giving rise to antibodies to the non-truncated protein. Examples of such truncated proteins include Sp1 comprising amino acids 115-127 of SEQ ID NO:2 and Sp2 comprising amino acids 1-279 of SEQ ID NO:2. Thus, the present invention further includes a method of raising immunoglobins comprising administering a protein of the present invention to a mammal, such as a rabbit or human, and optionally isolating the immunoglobins generated.
The present invention also provides nucleic acid encoding the protein of the present invention.
In particular, according to another aspect of the present invention there is provided nucleic acid comprising the sequence from positions 18 to 917 shown in SEQ ID NO: 1, or from positions 77 to 991 shown in SEQ ID NO:3.
The present invention also includes DNA which shows homology to the sequence of the invention. Typically homology is shown when 50% or more of the nucleotides are common, more typically 60% or 65%, preferably 70%, more preferably 75%, even more preferably 80% or 85%, especially preferred are 90%, 95%, 98% or 99% or more homology.
The present invention also includes DNA which hybridises to the DNA of the present invention. Preferably such DNA codes for at least part of an ABA signalling component.
The present invention also encompasses nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used a probe to identify similar coding sequences in other organisms etc.
The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
The term xe2x80x9ccomplementaryxe2x80x9d also covers nucleotide sequences that can hybridise to the nucleotide sequences of the coding sequence.
The term xe2x80x9cvariantxe2x80x9d also encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.
Preferably such hybridisation occurs at, or between, low and high stringency conditions. In general terms, low stringency conditions may be defined as 3xc3x97SSC at about ambient temperature to about 65xc2x0 C., and high stringency conditions as 0.1xc3x97SSC at about 65xc2x0 C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3xc3x97SSC is 3 times as strong as SSC and so on.
The present invention also comprises sequences obtained from PCR techniques using a primer derived from the sequence of the present invention.
The DNA of the invention may be cDNA or DNA which is in isolated form.
The invention further includes DNA which is degenerate as a result of the genetic code to the DNA of the present invention and which codes of a protein which is capable of affecting an ABA response, and/or responding to ABA.
The present invention also relates to vectors which comprise the nucleic acid of the present invention, host cells which are genetically engineered with the vectors of the present invention and the production of proteins of the present invention by recombinant techniques. Such system will be well known to those skilled in the art. The term xe2x80x9cvectorxe2x80x9d includes expression vectors and transformation vectors. The term xe2x80x9cexpression vectorxe2x80x9d means a construct capable of in vivo or in vitro expression.
Preferably the host cell is a plant, seed, fungus or mammalian cell. For the avoidance of doubt fungus includes yeast. Thus the present invention also provides suitably transformed plants, seeds, fungi and mammals.
The present invention therefore provides nucleic acid according to the present invention which is operably linked to a promoter. It is proposed that this system may be used in a method of selecting compounds capable of affecting a plant""s response to stress. Such a method may comprise screening compounds which bind to the expressed ABA signalling component and selecting compounds exhibiting said binding.
The invention thus further provides compounds selected using said screen and particularly their use as agrochemicals.
Agrochemical formulations are known and a skilled worker would readily be able to formulate an acceptable composition comprising an effective amount of the agrochemical.
A particularly preferred feature of the present invention is a plant or seed transformed with the nucleic acid of the present invention such that the ABA-signalling component of the present invention is expressed or rather over-expressed, in the plant or seed.
In such cases the promotor may be an inducible promotor. The advantage of using such a system is that expression can be controlled.
Such transformed plants, which may be monocotyledonous as well as dicotyledonous, may have improved growth and development over non-transformed plants. This may include reduced premature growth of buds and improved protection/tolerance against adverse environmental conditions such as drought, high salinity, cold temperature and frost. A particular feature may be improved tolerance to water stress. One way this may manifest itself is through reduced water loss. Plants will be expected to show reduced wilting.
The transformed seeds may have improved embryogenesis and better seed-storage protein formation compared to the wild-type. They may also show reduced premature germination and growth.
The method employed for transforming the host cells is not especially germane to this invention and any method suitable for the target host may be used. For example, transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation techniques are known from the literature such as agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, and microprojectile transformation.
Neither is the plant species into which the sequence is inserted particularly germane to the invention. Dicotyledonous and monocotyledonous plants can be, transformed. This invention may be applied to any plant for which transformation techniques are, or become, available. The present invention can therefore be used in a variety of plants, including field crops such as canola, sunflower, tobacco, sugarbeet, and conon; cereals such as wheat, barley, rice, maize and sorghum; fruit and vegetables. The invention is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues.
Mammalian homologs of the sequences derivable from plants may have application in therapy, and particularly in gene therapy. Thus according to a further aspect of the present invention there is provided pharmaceutical compositions comprising the protein and nucleic acid of the present invention and a pharmaceutically acceptable diluent, excipient, carrier or adjuvant.
The pharmaceutical composition may be for human or animal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular individual.
The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, adjuvant or excipient. The choice of pharmaceutical carrier, excipient, adjuvant or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise asxe2x80x94or in addition toxe2x80x94the carrier, excipient, adjuvant or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid delivery.
Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
The present invention also provides an anti-sense sequence and host cells further comprising an anti-sense sequence to the nucleic acid of the present invention. Again such sequences may be used in screening methods.